Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders. Transmembrane or otherwise tumor-associated polypeptides specifically expressed on the surface of cancer cells as compared to normal, non-cancerous cell(s) have been identified as cellular targets for cancer diagnosis and therapy with antibodies. Identification of such tumor-associated cell surface antigen polypeptides, i.e. tumor associated antigens (TAA), allows specific targeting of cancer cells for destruction via antibody-based therapies.
The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) Nature Biotechnology 23(9): 1137-1146; Payne, G. (2003) Cancer Cell 3:207-212; (Polakis, P. Arming antibodies for cancer therapy. Curr Opin Pharmacol 5, 382-387, 2005). Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al (ed.s), pp. 475-506). Efforts to improve the therapeutic index, i.e. maximal efficacy and minimal toxicity of ADC have focused on the selectivity of polyclonal (Rowland et al (1986) Cancer Immunol. Immunother., 21:183-87) and monoclonal antibodies (mAbs) as well as drug-linking and drug-releasing properties (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drug moieties used in antibody drug conjugates include bacterial protein toxins such as diphtheria toxin, plant protein toxins such as ricin, small molecules such as auristatins, geldanamycin (Mandler et al (2000) J. of the Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al (1986) supra). The drug moieties may affect cytotoxic and cytostatic mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
The auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin (WO 02/088172), have been conjugated as drug moieties to various antibodies (Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al (2003) Nature Biotechnology 21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465; US 2004/0018194; WO 04/032828; Mao et al (2004) Cancer Research 64(3):781-788); Bhaskar et al (2003) Cancer Res. 63:6387-6394); WO 03/043583; U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431; US 2005/0238649).
Conventional means of attaching, i.e. linking through covalent bonds, a drug moiety to an antibody generally leads to a heterogeneous mixture of molecules where the drug moieties are attached at a number of sites on the antibody. For example, cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody or through cysteine sulfhydryls (thiols) activated by reducing interchain disulfide bonds, generating a heterogeneous antibody-drug conjugate mixture, containing species with different molar ratios of drug to antibody, linked at different sites, each with a distinct in vivo profile of pharmacokinetics, efficacy, and safety (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Wang et al (2005) Protein Sci. 14:2436-2446). Depending on reaction conditions, the heterogeneous mixture typically contains a distribution of antibodies with from 0 to about 8, or more, attached drug moieties. In addition, within each subgroup of conjugates with a particular integer ratio of drug moieties to antibody, is a potentially heterogeneous mixture where the drug moiety is attached at various sites on the antibody. Analytical and preparative methods may be inadequate to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from a conjugation reaction. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Their reactivities with linker reagents and drug-linker intermediates are dependent on factors such as pH, concentration, salt concentration, and co-solvents. Furthermore, the multi-step conjugation process may be nonreproducible due to difficulties in controlling the reaction conditions and characterizing reactants and intermediates.
Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London, at page 55). Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups. Solvent-accessible inter-chain disulfide bond cysteines with serine to allow directed conjugation to the remaining cysteines (McDonagh et al (2006) Protein Eng. Des. Sel. 19:299-307). However, elimination of these disulfide bonds could disrupt quaternary structure of the antibody, thereby perturbing the behavior of the antibody in vivo, including changes in antibody effector functions (Michaelsen et al (1994) Proc Natl Acad Sci USA 91:9243-9247; Romans et al (1977) Proc Natl Acad Sci USA 74:2531-2535; Seegan et al (1979) Proc Natl Acad Sci USA 76:907-911). Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent attachments to ligands or to form new intramolecular disulfide bonds (Better et al (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al (1994) Bioconjugate Chem. 5:126-132; Greenwood et al (1994) Therapeutic Immunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci USA 96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214; Chmura et al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No. 6,248,564). However, engineering in cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free Cys) residues or those which are relatively accessible for reaction or oxidation. In concentrated solutions of the protein, whether in the periplasm of E. coli, culture supernatants, or partially or completely purified protein, unpaired Cys residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and hence protein dimers or multimers. Disulfide dimer formation renders the new Cys unreactive for conjugation to a drug, ligand, or other label. Furthermore, if the protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys and an existing Cys residue, both Cys thiol groups are unavailable for active site participation and interactions. Furthermore, the protein may be rendered inactive or non-specific, by misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).
Cysteine-engineered antibodies have been designed as FAB antibody fragments (thioFab) and expressed as full-length, IgG monoclonal (thioMab) antibodies (US 2007/0092940, the contents of which are incorporated by reference). ThioFab and ThioMab antibodies have been conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare antibody drug conjugates (Thio ADC).
MUC16 is a tumor associated antigen polypeptide, expressed by the human ocular surface epithelia (Argueso et al (2003) Investigative Ophthalmology & Visual Science 44(6):2487-95) in the mucosa of the bronchus, fallopian tube, and uterus (Kabawat et al. (1983) International Journal of Gynecological Pathology 2:275-85). One proposed function of MUC16 is to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces. Highly polymorphic, MUC16 is composed of three domains, a Ser-/Thr-rich N-terminal domain, a repeat domain of between eleven and more than 60 partially conserved tandem repeats of on average 156 amino acids each, and a C-terminal non-repeating domain containing a transmembrane sequence and a short cytoplasmic tail. MUC16 is heavily O-glycosylated and N-glycosylated (O'Brien et al (2002) Tumour Biol. 23:154-169; O'Brien et al (2001) Tumour Biol. 22:348-366 (2001); Fendrick et al (1997) Tumour Biol. 18:278-289; Wong et al (2003) J. Biol. Chem. 278:28619-28634; McLemore et al (2005) biol. Res. Nurs. 6:262-267). mRNA encoding the MUC16 polypeptide expressed from the MUC16 gene is significantly, reproducibly and detectably overexpressed in certain types of human cancerous ovarian, breast and pancreatic tumors as compared to the corresponding normal human ovarian, breast and pancreatic tissues, respectively (WO 2007/001851). A variety of independent and different types of cancerous human ovarian tissue samples quantitatively analyzed for MUC16 expression show the level of expression of MUC16 in the cancerous samples is variable, with a significant number of the cancerous samples showing an at least 6-fold (to as high as an about 580-fold) increase in MUC16 expression when compared to the mean level of MUC16 expression for the group of normal ovarian tissue samples analyzed. In particular, detectable and reproducible MUC16 overexpression was observed for ovarian cancer types; endometrioid adenocarcinoma, serous cystadenocarcinoma, including papillary and clear cell adenocarcinoma, as compared to normal ovarian tissue. Due to its overexpression in certain human tumors, the MUC16 polypeptide and the nucleic acid encoding that polypeptide are targets for quantitative and qualitative comparisons among various mammalian tissue samples. The unique expression profiles of MUC16 polypeptide, and the nucleic acid encoding that polypeptide, can be exploited for the diagnosis and therapeutic treatment of certain types of cancerous tumors in mammals.
CA125 (Carcinoma antigen 125 (O772P, CA-O772P, CA-125) is an extracellular shed protein encoded by the MUC16 gene (Yin et al (2002) Intl. J. of Cancer 98(5):737-740), and a serum marker used routinely to monitor patients with ovarian cancer. CA125 is a mullerian duct differentiation antigen that is overexpressed in epithelial ovarian cancer cells and secreted into the blood, although its expression is not entirely confined to ovarian cancer (Bast et al (1981) J. Clin. Invest. 68:1331-1337). Serum CA125 levels are elevated in about 80% of patients with epithelial ovarian cancer (EOC) but in less than 1% of healthy women (Bast et al. (1983) N. Engl. J. Med. 309:883-887). CA125 is a giant mucin-like glycoprotein present on the cell surface of tumor cells associated with beta-galactoside-binding, cell-surface lectins, which are components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression (Seelenmeyer et al (2003) Journal of Cell Science 116(7):1305-1318). High serum concentration of CA125 is typical of serous ovarian adenocarcinoma, whereas it is not elevated in mucinous ovarian cancer. CA125 is not recommended for ovarian cancer screening because normal level does not exclude tumor. However, CA125 detection is a standard tool in monitoring clinical course and disease status in patients who have histologically confirmed malignancies. Numerous studies have confirmed the usefulness of CA125 levels in monitoring the progress of patients with EOC (Bast et al (1998) Int. J. Biol. Markers 13:179-187; Verheijen et al (1999) Sem. Cancer Biol. 9:117-124; Menon et al (2000) Curr. Opin. Obstet. Gynecol. 12:39-42; Meyer et al (2000) Br. J. Cancer 82:1535-1538), and as a cancer serum marker. A rise in CA125 levels typically precedes clinical detection by about 3 months. During chemotherapy, changes in serum CA125 levels correlate with the course of the disease. CA125 is used as a surrogate marker for clinical response in trials of new drugs. On the other hand, CA125 is not useful in the initial diagnosis of EOC because of its elevation in a number of benign conditions (Bast et al (1998) Int. J. Biol. Markers 13:179-187; Meden et al (1998) Int. J. Biol. Markers 13:231-237). The CA125-specific antibody MAb-B43.13 (oregovomab, OvaRex MAb-B43.13) was in clinical trials for patients with ovarian carcinoma as an immunotherapeutic agent Mobus et al (2003) American Journal of Obstetrics and Gynecology 189(1):28-36; Ehlen et al (2005) International journal of gynecological cancer 15(6):1023-34.
Certain anti-MUC16 antibodies, including 3A5 and 11D10, have been disclosed in WO 2007/001851; U.S. Ser. No. 11/452,990, filed 14 Jun. 2006, Dennis et al, “Compositions and Methods for the Diagnosis and Treatment of Tumor”, the contents of which are incorporated by reference. The 3A5 monoclonal antibody binds multiple sites of the MUC16 polypeptide with 433 pM affinity by OVCAR-3Scatchard analysis. The 3A5 and 11D10 anti-MUC16 antibodies have been conjugated to auristatin drug moieties MMAE and MMAF. The conjugates inhibit in vitro tumor cell proliferation (WO 2007/001851). An 11D10 anti-MUC16 antibody was conjugated to the maytansinoid DM1 drug moiety (US 2005/0276812). Certain anti-MUC16 antibody variants have been cysteine engineered by the introduction of a cysteine amino acid unit and conjugated to DM1 (US 2007/0092940, the contents of which are incorporated by reference).